Abstract The tumor microenvironment is composed of highly heterogeneous niches, often with varying degrees of immune infiltration. The spatial distribution of immune cells with respect to malignant cells can directly impact patient prognosis and overall survival outcomes. The Visium CytAssist Spatial Gene Expression assay uses a whole transcriptome probe-based approach, termed RTL, to detect and quantify mRNA expression with spatial context. Although examination of the tumor microenvironment with an RTL-based spatial assay can provide significant transcriptomic information concerning regions of interest, immune cells frequently have extremely low mRNA expression levels and can be difficult to detect. The use of antibody-conjugated probes specific to immune cell epitopes, which are highly expressed, can enhance data recovered from these tumor samples, enabling spatially accurate detection of immune populations. The Visium CytAssist Spatial Proteogenomic Solution enables identification of immune-specific epitopes via antibody-conjugated probes from the same tissue slide used for transcriptomic analysis. Using the CytAssist workflow, we showcase the ability to comprehensively resolve immune cells associated with multiple immune and tumor tissues, including an array of human breast cancer punches. Spatial expression patterns of immune markers map back to distinct morphological features within the samples, allowing identification of differentially-expressed genes associated with those areas. Overall, these data highlight the value of Visium CytAssist Spatial Proteogenomic Solution in immuno-oncology studies, through the integration of spatially resolved transcriptomic and immune cell marker data.